Figure 3
From: A cell cycle-independent mode of the Rad9-Dpb11 interaction is induced by DNA damage
Dot1 is required for phosphorylation of Rad9 S/TP sites and interaction with Dpb11. (A) Dot1 is required for Rad9 association with a double strand break (DSB). Induction of an non-repairable DSB at MAT locus using galactose-induced HO. ChIP against Rad93FLAG to regions from 1.1 kb to 8 kb distal of the DSB site and 1, 2 and 4 h after DSB induction. FACS profiles in Fig. S3A. (B–D) The ‘histone pathway’ is required for efficient damage-induced phosphorylation of Rad9-T474 and binding to Dpb11. (B) Phleomycin-induced T474 phosphorylation is reduced in dot1Δ or rad9-Y789Q cells (deficient in TUDOR domain-dependent binding to K79-methylated H3). Experiment as in Fig. 2A, but with WT, dot1Δ and rad9-Y789Q cells. Pgk1 immunoblot serves as loading control. An asterisk denotes a crossreactive band. FACS profiles in Fig. S3D. (C) Dpb11 does not bind to Rad9 from extracts of G1-arrested, phleomycin-treated dot1Δ cells. GST-Dpb11-N pulldown as in Fig. 1A (D) DNA damage-induced Rad9-T474 phosphorylation in G1 as in (B), but with WT, ddc1-T602A, dot1Δ or dot1Δ ddc1-T602A strains. FACS profiles in Fig. S3E.
